Method for the preparation of a pancreatic heparinoid



P. BIANCHINI May 4, 1965 METHOD FOR THE PREPARATION OF PANCREATIC HEPARINOID Filed May 25, 1962 5 Sheets-Sheet 1 FIG. 1 6

FIG. 1a

FIG. 1d

INVENTOR. 8'3 170 5 1 62 ire/$171 BY {m 19%;, 77422:

"-0- B .'..A.. A f 7.3701 e 73;

May 4, 1965 P. BIANCHINI 3,131,996

METHOD FOR THE PREPARATION OF A PANCREATIC HEPARINOID Filed May 25, 1962 5 Sheets-Sheet 2 CAKE OF PANCREAS V DEFAT FOR 8 HOURS ACETONE DRY UNDER VACUUM DRY, DE FATTED PANCREAS AGITATE FOR I/2 HOUR ALKALINE SOLUTION AT 65-75C TO pH ABOUT 9.0

DISPERSION OF DEFATTED PANCREAS FILTER VPP ALKALINE SOLUTION 'AGITATE FOR 1/2 HOUR TO pH ABOUT 9.0

AT 6575C FILTRATE FILTRATE V COMBINED FILTRATES CONCENTRATE TO I/4 INITIAL VOLUME DIALYZE 24-72 HOURS ACETONE AND ACETIC ACID TO pH ABOUT 5.0

FILTER INVENT OR FIG. 26%

ORNEYS y 4, 1965 P. BIANCHINI 3,181,996

METHOD FOR THE PREPARATION OF A PANCREATIQ HEPARINOID Filed May 25, 1962 5 Sheets-Sheet 3 RAW PANCREATIC HEPARI NOID ENZYME AS PAPAIN DISSOLVE eoc, IHOUR AND ALKALINE SOLUTION TO pH ABOUT 8.0

DIALYZE 24-72 Hou|- FILTER ACETONE AND ACETIC ACID FILTRATE E T0 pH ABOUT 5.0

l DRY pp. i

ALCOHOL- ETHER DRIED ppt.

PURIFY SOLID I PHENOL PURIFIED PANCREATIC HEPARINOID FlG.2b.

INVENTOR Pietro Bicmchini ORNEYS' May 4, 1965 METHOD FOR THE PREPARATION OF A PANCREAI'IC HEPARINOID Filed May 25, 1962 P. BIANCHINI 3,181,996

5 Sheets-Sheet 4 INVENTOR. )Qefi-o 16/ an 7? 1 May 4, 1965 PLBIANCHINI 3,181,996

METHOD FOR THE PREPARATION OF A PANCREATIC HEPARINOID Filed May 25, 1962 5 Sheets-Sheet 5 INVENTOR. 1 /1 5/4 7 c A1 77;

United States Patent METHQD 1 6R THE PREPARATION 6F A PANQREATKC HEPARINOID Pietro Bianchini, Como, Italy, assignor to Crinos industria Farmacobiologica S.p.A., Villaguardia, Italy Filed May 25, 1962, Ser. No. 197,772

Claims priority, application Italy, May 25, 1961,

9,644/61 8 @laims. (Cl. 167-74) The present invention relates to a method for the preparation of a pancreatic heparinoid.

The pancreatic heparinoid (Hepd. P) is a substance of a polyanionic nature, belonging to the group of complex sulphomucopolysaccharides, on the basis of its content of hexosamines (350 ,ug/rng), hexuronic acids (360 ,ug./ mg), and sulphonic groups.

According to this invention there is provided a method for the preparation of a pancreatic heparinoid which comprises:

(a) Removing the fat from a cake of pancreas derived from the lipocaic process or derived from fresh pancreas by subjecting the cake to the action of an organic solvent;

(b) Then drying the cake after separation from the organic solvent extract;

(c) Dispersing the resultant powdered cake in an aqueous alkaline solution;

(d) Agitating the resultant dispersion at an elevated temperature and filtering while hot;

(e) Dispersing the resultant precipitate in an aqueous alkaline solution and agitating the resultant dispersion at an elevated temperature and filtering while hot;

(1) Combining the alkaline filtrates from stages (d) and (e) and concentrating the combined alkaline filtrates to a substantially smaller volume than the initial volume;

(g) Dialyzing the concentrate from stage (1) and if necessary filtering to remove any insoluble substances present;

(11) Precipitating the liquid with a suitable organic solvent in the presence of a weak acid;

(1) Drying the resultant precipitate and dissolving it in water with addition of a small amount of a suitable protein-digesting enzyme and rendering the mixture weakly alkaline by addition of alkali;

(j) Dialyzing the solution from stage (i) and filter- (k) Precipitating the filtrate from stage (j) with a suitable organic solvent in the presence of a weak acid;

(I) Drying the precipitate with a suitable organic solvent; and

(m) Optionally purifying the dried precipitate from stage (I) with a suitable organic liquid in which the heparinoid is insoluble.

As mentioned above, the starting material may be a cake of pancreas derived from the lipocaic process or derived from fresh pancreas. In the latter case the following rocedure may be employed prior to carrying out a process embodying the steps (a) to (m) of this invention: grinding of the fresh organ, homogenization by boiling with dilute hydrochloric acid for the required period, neutralization with an alkali metal hydroxide, and then allowing the mixture to stand. The pH of the liquid is then made weakly acidic with acetic acid, the cake obtained by means of filtration is then dried under vacuum at a maximum temperature of 50 C.

In carrying out a process. embodying this invention one or more of the following advantageous procedures may be adopted.

The aqueous alkaline solution of one or both of the features (c) and (e) may be arranged to be at a pH of about 9.

The elevated temperature of one or both the features (d) and (e) may be Within the range of about 65 to C.

The agitation operation of one or both of the features (01) and (e) may be continued for at least half an hour.

The filtration operation of one or both of features (d) and (e) may be carried out with a pressure filter. Also the filtration operation of feature (j) may be carried out with a pressure filter.

The combined alkaline filtrates of feature (f) may be concentrated to about a quarter of the initial volume.

With regard to both or either of features (g) and (j), the dialyzing procedure may be effected by means of a semipermeable membrane, e.g. against running water, for a period such as for instance 24 to 72 hours.

The solvent of feature (a), (h) or (k) or any two or all three of the features (a), (h) and (k) may comprise a ketone, e.g. acetone.

1 part by weight of the resultant precipitate of feature (1') may be dissolved in 5 to 15 parts by volume of water. Also 1 part by weight of the resultant powdered cake of feature (0) may be dispersed in 5 to 15 parts by volume of the aqueous solution.

The protein-digesting enzyme of feature (i) may comprise papain used in the presence of a reducing agent; the reducing agent may for instance comprise sodium hyposulphite or cysteine. The protein-digesting enzyme of feature (1') may comprise trypsin which may be used in the absence of a reducing agent. A mixture of papain and trypsin may be used, if desired.

The mixture of feature (i) may be rendered weakly alkaline to a pH of about 8 by addition of the required amount of alkali. After the solution of feature (i) has been rendered weakly alkaline, the solution may be maintained at an elevated temperature, e.g. about 60 C., for at least an hour.

The solvent of feature (I) may comprise a mixture of alcohol and ether.

The organic liquid of feature (m) may comprise a phenol, e.g. phenol itself.

The precipitation of feature (It) or feature (k) or both features (h) and (k) may be carried out at a pH of about 5.

The pancreatic heparinoid obtained by a process embodying this invention gives typical reactions with many dyes and basic substances, such as Azure A (with which it forms metachromatic complexes), trypafiavin and rivanol (with which it forms insoluble and inactive complexes), long-chain quaternary ammonium bases, octylamine and clupeine (with which if forms easily dissociable complexes).

FIGS. 2a and 212 show an illustrative flow diagram of the process of the present invention.

An example of highly specific activity in the formation of complexes with trypafiavin, rivanol, and toluidine blue is shown in FIGURES 1a, 1b, 1c and 1d of the accompanying drawings, in which certain natural substances with a similar structure are compared (heparin, FIG. 1a; pancreatic heparinoid, FIG. lb; duodenal heparinoid, FIG. 10; controitin sulphate, FIG. 1d).

Total cholesterol, mg./100 ml.

the three curves F, G and H refer to the total plasma fatty acids of The 1 2 and 3 of the diagram of FIG. 4 refer,

inoleic and The chromtography was performed on It is observed that the stearate group remains un- The pancreatic heparinoid apparently is not an antirinoid exer- Lipemia, mg./100 ml.

ic heparinoid, and the ings,

ith 5% mineral oil ed of 90% acetic The lipidic spectra are revealed by Nile Ble/NaOH.

Dose, mgJkg.

ography of the blood fatty acids idic spectrum in the animals treat t with heparinoid results in a definite normalization of the lipidic picture.

the stearic acid group, the group of hi h dosages. to clinical activity, it is interesting to 0bing: Since the pancreatic hepa cises notable lipid-regulatory activity on the blood (even the experimental lipodysproteinemia induced by Triton ct al., as can be observed in the following table:

Substance Controls (C T-l-Hepd. P. I+Heparin The paper chromat presents a different lip Treatmen In FIGURE 4 of the accompanying draw rve P refers to the controls, curve G refers to and curve H refers to Triton+P Hepd.

abscissae tively, to:

d oleic acids, and the group of l with a migrating solvent compos however the oleate and linoleate groups are notably influenced. These data are confirmed by gaschromatographic analysis.

even in In reference serve the follow' when administered by mouth), clinical tests have been ed with Triton, Triton-i-pancreat controls.

rats; cu

Triton,

respec palmitic an myristic acids.

Whatman paper 3 MM pretreated w and,

acid

changed coagulant,

Velocity in Heparin equiv.

d dendritic y, it is observed that ring factor ls (either intravenously or is less S m ms 0 mm .m H

eatic heparinoid separates a and b), which are probably artifacts due to the formation of two different complexes s.5 10- 5.s 10 4.s 10

ment also, the pancreatic 25 W0 components, with R Hep. (which is taken as id has been isolated in the able to liberate the clea cted into anima traperitoneally), although the quantity liberated than that liberated by heparin.

The curves A, B and C of the diagrams shown in the above-mentioned figures pertain, respectively, to

A trypafiavin B rivanol;

C toluidine blue.

tion occurs.

Substance H 8.6-Borate buffered soln.-T. 20 C.1O V/cm./3h

In the chromatographic treat rinoid (Hepd. P) presents t larger than that of the heparin, the reference substance).

The pancreatic heparino crystalline state, in the form of rosette an structures.

Considering the biological activit The clearing activity has been deter the measurement of the optical density In electrophoresis the pancr to two components with the buffered borate solution, by means of which the 10 migra Heparin Hepd. P (a) Hcpd. P

Whatman paper hepa the heparinoid is when it tituted, administering composed of 10 mg. of a standard 2:1 mixtures of plasma of 200 gm. rats and a solution of 0.25% Ediol. respectively; the measurement performed 10 minutes after intravenous injection of hepar FIGURE 3 of the accompanying drawings shows the course of the clearing action of the pancreatic heparinoid (Hepd. P) in relation to heparin (Hep).

Daily 7 741 2970 G RZZATA K5A 3G5 14 680 263495848 amld lnmlfiwumru Albumin/ Beta/ (1050 in glob- Alpha tablets Globulin Alpha,

ls treated with Triton.

anima The preliminary results obtained in a group of hypertable cholesterolemic subjects are reported in the following TABLE optic I 5 9 mm 3993 72 9 583351 2 G3 G0 1 h 3 54 4444 &7 73 5&3 3 5 5 43 2 33 22353 &6& Dm

H0 9. 0&7 255790060 1115012006907 5 605 m 83019485225 159349 62 1752092239 mwm ww 7 7 7 7 fim7 6 7 7 7 7 7 7 6 7 7 G76 7 7 677 177 9206 Tmm Pe n n n n h n 7 7575229753 m m m m m m m 4 454 55 54 C n u u n n n une mmmmmm m m mmmmmmmmmm "5 m TITTTT r. I r rffrffrf afi 000000 0 0 0000000000 wm NNNNNN N N NNNNNNNNNN Choles- Plasma Hemoterolemia, g. percent In addition, the pancreatic heparinoid possesses an intense lipid-regulatory activity, in the sense that it corrects (8) V.S., a. 40 Chronic hepatitis. (9) M a Arterial hypertension atherosclerosis.

TABLE Choles- Plasma Hemo- Vis- Total Globulin Albu- Daily terolopt c coagucosity, protein- Albumin/ Beta/ dose in emra, g. density lation centiemia, g. min, glob- Alpha tablets percent (600 m time poise percent percent Alphai, Alpha, Beta, Gamma, ulin percent percent percent percent Arterial hypertension 210 118 Norm. 4. 6 7. 25 59. 9 2. 3 5. 9 11. 2 20. 7 1. 50 4 4 atherosclerosis myocardiosclcrosis. (1 t) T.V., a. 56 300 133 Norm. 6. 2 7.8 63. 5 1.8 9.2 11.8 13.6 1. 74 3. 54 Atherosclerosis hypcrtcn- 285 129 Norm. 5. 5 7. 5 58. 2 2. 4 8. 3 13. 1 18 1. 39 2. 93 5 sum. (15) M.P., a. 77.- 300 134 Norm. 5. 5 7. 5 6G 1. 7 5. 3 12 14. 4 1. 94 7. 33 Ather0scler0sis 256 G Norm. 5 7. 2 50 2 6 15 27 1 4. 03 4 6) 1.15., a. 55 256 120 Norm. 4. 5 6. 4 57 1.8 5. 4 11. 6 24. 2 1. 3 2. 31 Atherosclerosis retinal 300 119 NOIIIL 4. 8 7 53 3. 5 7 12 24. 5 l. 1 3. 5 5

spasm hypertension.

A method of preparation a pancreatic heparin'oid, embodying this invention, will be described in detail by the ttollowing examples.

Example 1 50 kg. of caked pancreas (from the residue of the lipocaic process) are freed from fat, continually for 8 hours in an appropriate apparatus, using 200 litres of acetone. After separation from the acetonic liquid, the cake is dried in an oven under vacuum, and the powder so obtained is dispersed in water (alkalinized up to a pH of 9 using caustic soda) in a proportion of 1 part by weight of powder to 10 parts by volume of water. While shaking, the suspension is brought up to a temperature of 70 C., maintaining a constant pH for minutes. After filtration of the hot contents (using a pressure filter), the procedure of dispersion in alkaline water is repeated. The combined alkaline liquids are concentrated until the volume is reduced to A of the initial volume, and the concentrated alkaline liquid is dialyzed through a semi-permeable membrane for 48 hours against running water. After filtration (to remove a light precipitate that is formed), precipitation is effected wit-h 1.25 volumes of acetone, while shaking vigorously; at the same time the pH is brought to 5 with acetic acid. After drying, the precipitate so obtained is dissolved in water as a 10% solution, with the addition of 1% sodium hyposulphite and 0.1% of commercial papain. The pH is brought to 8 using caustic soda, and the liquid is maintained at 60 C. for 8 hours. The contents are then dialyzed against running water for 48 hours through semipermeable membranes, the contents are filtered (using a pressure filter), the clear filtrate is reprecipitated with 1.5 volumes of acetone, and the pH is again brought to 5 with acetic acid. The obtained precipitate is dehydrated by means of washing with alcohol-ether, and the white powder so obtained can be used directly for the preparation of tablets. It can be purified by means of treating it with 90% phenol (5% of powder by weight per unit volume of phenol). The product, insoluble in phenol, has a grade of purity 10 times that of the crude product.

Example 2 For extraction from fresh pancreas, the fresh organ is finely ground, 3 volumes of 5% hydrochloric acid are added and the contents are homogenized and boiled for 10 minutes; the pH is then brought to 6.8-7 with caustic soda, the contents are left to stand for two hours and then are re-acidified with acetic acid (pH 5). After filtration and drying under vacuum (at a maximum temperature of 50 C.) the cake is ready for the extraction of the heparinoid. The heparinoid is then prepared as described in Example 1.

What I claim is:

1. A method for the preparation of a pancreatic heparinoid which comprises:

(at) removing the fat from a cake of pancreas by subjecting the cake to the action of an organic solvent;

(b) then drying the cake after separation from the organic solvent extract;

(0) dispersing the resultant powdered cake in an aqueous alkaline solution;

(d) agitating the resultant dispersion at an elevated temperature and filtering while hot;

(e) dispersing the resultant precipitate in an aqueous alkaline solution and agitating the resultant dispersion at an elevated temperature and filtering while hot;

(f) combining the alkaline filtrates from stages (d) and (e) and concentrating the combined alkaline filtrates to a substantially smaller volume than the initial volume;

(g) dialyzing the concentrate from stage (7);

(h) precipitating the liquid with a suitable organic solvent in the presence of a weak acid;

(i) drying the resultant precipitate and dissolving it in water with addition of a small amount of a suitable protein-digesting enzyme and rendering the mixture weakly alkaline;

(j) dialyzing the solution from stage (i) and filtering;

(k) precipitating the filtrate from stage (i) with a suitable organic solvent in the presence of a weak acid;

(I) and drying the precipitate with a suitable organic solvent.

2. Method according to claim 1, further comprising filtering the liquid from the stage (g) to remove insoluble substance prior to precipitating the liquid according to stage (/1); and purifying the dried precipitate from stage (I) with a suitable organic liquid in which the heparinoid is insoluble.

3. Pancreatic heparinoid prepared by the method of claim 1.

4. A method for the preparation of a pancreatic heparinoid which comprises:

(a) removing the fat from a cake of pancreas by sub}ecting the cake to the action of an organic solvent;

(1)) then drying the cake after separation from the organic solvent extract;

(c) dispersing each part by weight of the resultant powdered cake in an aqueous alkaline solution at a pH of about 9;

(d) agitating for at least half an hour the resultant dispersion at an elevated temperature within the range of about 65 to 75 C., and filtering while hot;

(e) dispersing the resultant precipitate in an aqueous alkaline solution at a pH of about 9, and agitating the resultant dispersion for at least half an hour at an elevated temperature within the range of about 65 to 75 C., and filtering while hot;

(f) combining the alkaline filtrates from stages (d) and (e) and concentrating the combined alkaline filtrates;

(g) dialyzing the concentrate from stage through a semi-permeable membrane for a period within the range of 24 to 72 hours;

7 (h) precipitating the liquid with a suitable organic solvent in the presence of a weak acid; (i) drying the resultant precipitate and dissolving each part by weight of it in water with addition of a small amount of a suitable protein-digesting enzyme and bringing the mixture to a pH of about 8 by addition of alkali, the solution being maintained at a temperature of about 60 C. for at least one hour;

(j) dialyzing the solution from stage (i) through a semi-permeable membrane for a period within the range from 24 to 72 hours, and filtering;

(k) precipitating the filtrate from stage (j) with a suitable organic solvent in the presence of a weak acid;

(I) drying the precipitate with a mixture of alcohol and ether;

(m) and purifying the dried precipitate from stage (I) with a phenol in which the heparinoid is insoluble.

5. Method according to claim 4 wherein the organic solvent of stages (a), (h) and (k) includes a ketone. V

6. Method according to claim 4, wherein the proteindigesting enzyme of stage (i) comprises papain used in the presence of a reducing agent selected from the group consisting of sodium hyposulphite and cystein.

7. Method according to claim 4, wherein the proteindigesting enzyme of stage (i) comprises trypsin.

8. Method according to claim 4 wherein the precipitation of stages (11) and (k) is carried out at a pH of about 5.

References Cited by the Examiner UNITED STATES PATENTS 2,954,321 9/60 Coleman et al 16774 JULIAN S. LEVITT, Primary Examiner.

FRANK CACCIAPAGLIA, 1a., LEWIS GOTTS,

Examiners. 

1. A METHOD FOR THE PREPARATION OF A PANCREATIC HEPARINOID WHICH COMPRISES: (A) REMOVING THE FAT FROM A CAKE OF PANCREAS BY SUBJECTING THE CAKE TO THE ACTION OF AN ORGANIC SOLVENT; (B) THEN DRYING THE CAKE AFTER SEPARATION FROM THE ORGANIC SOLVENT EXTRACT; (C) DISPERSING THE RESULTANT POWDERED CAKE IN AN AQUEOUS ALKALINE SOLUTION; (D) AGITATING THE RESULTANT DISPERSION AT AN ELEVATED TEMPERATURE AND FILTERING WHILE HOT; (E) DISPERSING THE RESULTANT PRECIPITATE IN AN AQUEOUS ALKALINE SOLUTION AND AGITATING THE RESULTANT DISPERSION AT AN ELEVATED TEMPERATURE AND FILTERING WHILE HOT; (F) COMBINING THE ALKALINE FILTRATES FROM STAGES (D) AND (E) AND CONCENTRATING THE COMBINED ALKALINE FILTRATES TO A SUBSTANTIALLY SMALLER VOLUME THAN THE INITIAL VOLUME; (G) DIALYZING THE CONCENTRATE FROM STAGE (F); (H) PRECIPITATING THE LIQUID WITH A SUITABLE ORGANIC SOLVENT IN THE PRESENCE OF A WEAK ACID; 